铜绿假单胞菌噬菌体PaP1的基因组学研究：一种由EndonucleaseⅤ介导的细菌免疫新机制的发现与证实
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摘要
铜绿假单胞菌是临床上常见的革兰阴性条件致病菌，能耐受多种抗生素，是医院内感染的主要病原菌之一。噬菌体是一类专门侵染细菌的病毒，在自然界中广泛存在，数目约为细菌的10倍。噬菌体易于培养、基因组小且结构简单，一直是研究多种基本生命现象的重要对象与材料。对噬菌体进行鉴定和分类是噬菌体研究中的一项基本工作。目前大部分新鉴定的噬菌体已经归类为不同的属，但还有相当一部分噬菌体没有明确的分类。
对噬菌体进行全基因组测序和注释是做噬菌体研究的重要内容。最初的基因组测序主要使用全基因组“鸟枪”测序法。然而某些噬菌体基因组的“鸟枪”测序并不顺利，有时甚至不得不半途而废。早在2004年，本实验室就遇到了一个难以测序的铜绿假单胞菌噬菌体PaP1的基因组。当时张克彬博士和黄建军博士前后分两次委托不同的商业化测序公司采用当时流行的“鸟枪”测序法对该噬菌体进行全基因组测序，然而始终得不到PaP1基因组的完整序列。因此噬菌体PaP1基因组的测序工作不得不搁置。
直到在2008年第二代测序技术的推广应用后，我们才委托测序公司通过罗氏454测序技术顺利地获得了噬菌体PaP1的全基因组序列（91,715bp）。本文将进行噬菌体PaP1基因组的详细注释与比较基因组学分析，并深入探讨该基因组“鸟枪法”难以测序的原因。研究内容和结果主要包括以下几个方面：
1.铜绿假单胞菌PA1及其噬菌体PaP1一般生物学特性的补充研究。确定了铜绿假单胞菌PA1的生长特点、革兰阴性形态及其胞外基质与鞭毛的形态。使用PEG8000浓缩沉淀的方法制备PaP1的粗制颗粒，再进行离心超滤得到纯化的PaP1颗粒。使用透射电镜观察PaP1颗粒的形态，分析出其具有二十面体对称的头部（直径约为69nm）和收缩性的尾部（长度约为139nm），这是确定其为肌尾噬菌体的重要依据。
2.噬菌体PaP1基因组末端的鉴定。使用罗氏454测序方法测出PaP1基因组全长为91,715bp。通过限制性酶切的方法确定PaP1基因组为线性dsDNA分子，并确定了其末端的位置。分别回收PaP1基因组DNA5’末端和3’末端的酶切片段，使用我们所设计的末端鉴定引物进行末端测序。我们设计了一种新的噬菌体末端鉴定策略，使用该策略鉴定出PaP1基因组DNA具有1190bp长的末端冗余。
3.噬菌体PaP1基因组的生物信息学及比较基因组学分析。通过GC含量的分析确定PaP1基因组的复制起始位点位于该基因组末端。通过开放阅读框（ORF）和编码序列（CDS）的鉴定确定了PaP1基因组有169个推定的基因（其中包含12个tRNA基因）。使用BlastP分析了PaP1基因的推定功能，并将PaP1基因组的注释信息提交给GenBank（登录号：NC_019913）。对铜绿假单胞菌的四株肌尾噬菌体（PaP1、JG004、PAK_P1和vB_PaeM_C2-10_Ab1）进行了比较基因组学分析，构建了其主要衣壳蛋白的进化树，并建立了噬菌体的一个新属：PaP1样噬菌体属。
4.噬菌体PaP1结构蛋白的SDS-PAGE及质谱鉴定。采用浓缩后的PaP1粗制颗粒进行SDS-PAGE实验，将PaP1的结构蛋白显示在聚丙烯酰胺凝胶上。分别切离不同的PaP1蛋白条带，并进行蛋白的胶内酶解，然后进行HPLC-MS。最后一共鉴定出12个PaP1结构蛋白的编码基因，即通过实验证实了这12个PaP1基因的功能是编码结构蛋白。
5.噬菌体PaP1基因组“鸟枪法”测序难题的研究。把之前PaP1基因组“鸟枪法”测序所获得的43个重叠群（contigs）定位在PaP1全基因组上，发现有约一半的序列是“鸟枪法”所测不出来的。我们阅读文献发现“鸟枪法”测序的宿主菌（大肠杆菌DH5α）可以编码识别修饰碱基的酶EndonucleaseⅤ（EndoⅤ）。而PaP1基因组DNA可以被EndoⅤ所切割，同样条件下“鸟枪法”测序成功的PaP3基因组DNA不可以被EndoⅤ所切割。这为PaP1基因组“鸟枪法”测序难题的解开提供了线索。
6. EndoⅤ介导的细菌免疫机制的研究。我们通过λ-Red重组的方法敲除了大肠杆菌DH5α编码EndoⅤ的基因nfi，构建了突变株DH5α Δnfi。然后使用DH5α Δnfi作为“鸟枪法”克隆建库的宿主菌，重新对PaP1基因组进行“鸟枪法”测序，结果顺利完成了其测序。我们通过单分子实时（SMRT）测序的方法鉴定出PaP1基因组DNA中分布有7557个修饰碱基（包括152个m4C和51个m6A）。据此我们提出细菌通过EndoⅤ排斥噬菌体含有修饰碱基核酸的这样一种新的细菌免疫机制。
综上所述，本文对PaP1长91,715bp的基因组进行了详细的注释，通过比较基因组学的分析建立了铜绿假单胞菌肌尾噬菌体中的一个新的属：PaP1样噬菌体属，解析了PaP1的12个结构蛋白编码基因，探讨了“鸟枪”测序法无法测序的PaP1基因组采用二代测序技术就可以测出全序列的根本原因并提供了解决办法，还揭示了细菌通过EndoⅤ免疫排斥外来侵染核酸、保持物种遗传稳定的免疫新机制。
Pseudomonas aeruginosa is a gram-negative opportunistic pathogen, frequently foundin clinical treatment and resistant to many kinds of antibiotics. Pseudomonas aeruginosa isone of the main pathogens in hospital infection. Phages are viruses specifically infectingbacteria and ubiquitous in the biosphere. Estimations of phage numbers are approximatelytenfold higher than those of bacteria. Phages are structurally simple, gnomically small andeasily cultured, making them constantly become important objects and materials of manybasic vital phenomena. Identification and classification of phages are groundwork in phagestudies. Currently, most of the newly identified phages have been categorized into differentgenera; whereas there are still some phages remain unclassified.
Whole genome sequencing and annotation are important work in phage studies.Shot-gun sequencing method had been the most popular genome sequencing method for along time. Some phages could not be successfully sequenced by shot-gun strategy or thesequencing process had to stop halfway. Early in the year2004, our laboratory encounteredPseudomonas aeruginosa phage PaP1with its genome hard to sequence. At that time,research students of our laboratory sent the PaP1genome DNA to a commercial sequencingcompany to have it sequenced by shot-gun strategy, but it failed. Then they sent it toanother commercial sequencing company to have it sequenced by shot-gun strategy, itfailed again. So the sequencing of PaP1genome has to be postponed.
In the year2008, the second generation sequencing method had spread worldwide. Wesent the PaP1genome DNA to a commercial sequencing company to have it sequenced byRoche/454sequencer. It gives us a whole PaP1genome sequence with a length of91,715bp. In this thesis, we will fully annotate the PaP1genome, perform comparative genomicanalysis of this genome, and extensively analyze the reason behind the PaP1genome hard to sequence by shot-gun sequencing method. The results are as follows:
1. Additional analysis of the common bionomics of Pseudomonas aeruginosa PA1andphage PaP1. We observed the growth characteristics, gram-negative morphology,extracellular matrix and flagella of Pseudomonas aeruginosa PA1. The PaP1particles wereconcentrated by using PEG8000, and purified by ultrafiltration. The morphology of PaP1was observed by transmission electron microscope. The result shows that PaP1have anicoschedra head~69nm in diameter and a contractile tail~139nm in length. This is animportant evidence for that PaP1is a member of myovirus.
2. Identification of the terminal redundancy of phage PaP1. Roche/454sequencingmethod reveals a whole PaP1genome sequence91,715bp in length. The linear dsDNAfeature and the location of the PaP1genome ends were characterized by restriction enzymedigestion analysis. We recovered the3’ and5’ end fragments of the PaP1genome, and thenterminal run-off sequencing of the two terminal fragments were carried out to identify theterminal sequence of the PaP1genome. We designed a new strategy to explore the terminalsequence of the PaP1DNA, and the result shows that the PaP1genome has1190bpterminally redundant regions.
3. Bioinformatic and comparative genomic analyses of the PaP1genome. GC skewanalysis reveals that the replication origin of the PaP1genome locates within the terminallyredundant region. The PaP1genome has169putative genes (including12tRNA genes),based on the analysis of open reading frames and coding sequences. BlastP analysis revealsputative functions of these genes. Both the genome sequence and the annotationinformations were submitted to GenBank under the accession number: NC_019913.Comparative genomic analyses of four Pseudomonas aeruginosa myovirus (PaP1, JG004,PAK_P1and vB_PaeM_C2-10_Ab1) were performed. We constructed the phylogenetic treeof these four phages and proposed a new genus: PaP1-like phages.
4. Identification of phage PaP1structural proteins. SDS-PAGE was used to visualizeeach structural protein of phage PaP1in the gel. At least17proteins with molecular weightsranging from6kDa to80kDa were resolved. Each protein band was then excised forHPLC-MS, permitting the allocation of15protein bands to12corresponding PaP1genes.
5. Analysis of the puzzle of the PaP1genome hard to sequence by shot-gun strategy.Shot-gun sequencing of the PaP1genome only gives us43contigs. We located these contigs to the PaP1genome sequence revealed by Roche/454sequencer and found thatapproximately half of PaP1genome can not be sequenced by shot-gun strategy. Then wefound that EndonucleaseⅤ（EndoⅤ）, produced by E. coli DH5α (the host bacteria ofshot-gun sequencing), can digest the PaP1genome DNA. But with the same condition, thePaP3genome DNA (successfully sequenced by shot-gun strategy) can not be digested byEndo Ⅴ. This result provides a hint for the puzzle of the shot-gun sequencing of the PaP1genome.
6. Analysis of EndoⅤ mediated immunity of bacteria. We knocked out the EndoⅤcoding gene (nfi) of E. coli DH5α using λ-Red recombination system and the recombinantstrain was named E. coli DH5α Δnfi. Then shot-gun sequencing of the PaP1genome DNAwas successfully completed using E. coli DH5α Δnfi as the host bacterium. SMRTsequencing revealed7557modified bases (including152m4C and51m6A) in the PaP1genome. These results indicate that the well-known DNA repair protein EndoⅤ ofE. colican mediate the immunity of modified bases containing invasive phage DNA.
In conclusion, this work fully annotated the PaP1genome, constructed a new genusnamed “PaP1-like phages” within the Myoviridae family, identified12PaP1structuralprotein coding genes, and reveal a new immunity mechanism for bacteria to resist phageDNA.
对噬菌体进行全基因组测序和注释是做噬菌体研究的重要内容。最初的基因组测序主要使用全基因组“鸟枪”测序法。然而某些噬菌体基因组的“鸟枪”测序并不顺利，有时甚至不得不半途而废。早在2004年，本实验室就遇到了一个难以测序的铜绿假单胞菌噬菌体PaP1的基因组。当时张克彬博士和黄建军博士前后分两次委托不同的商业化测序公司采用当时流行的“鸟枪”测序法对该噬菌体进行全基因组测序，然而始终得不到PaP1基因组的完整序列。因此噬菌体PaP1基因组的测序工作不得不搁置。
直到在2008年第二代测序技术的推广应用后，我们才委托测序公司通过罗氏454测序技术顺利地获得了噬菌体PaP1的全基因组序列（91,715bp）。本文将进行噬菌体PaP1基因组的详细注释与比较基因组学分析，并深入探讨该基因组“鸟枪法”难以测序的原因。研究内容和结果主要包括以下几个方面：
1.铜绿假单胞菌PA1及其噬菌体PaP1一般生物学特性的补充研究。确定了铜绿假单胞菌PA1的生长特点、革兰阴性形态及其胞外基质与鞭毛的形态。使用PEG8000浓缩沉淀的方法制备PaP1的粗制颗粒，再进行离心超滤得到纯化的PaP1颗粒。使用透射电镜观察PaP1颗粒的形态，分析出其具有二十面体对称的头部（直径约为69nm）和收缩性的尾部（长度约为139nm），这是确定其为肌尾噬菌体的重要依据。
2.噬菌体PaP1基因组末端的鉴定。使用罗氏454测序方法测出PaP1基因组全长为91,715bp。通过限制性酶切的方法确定PaP1基因组为线性dsDNA分子，并确定了其末端的位置。分别回收PaP1基因组DNA5’末端和3’末端的酶切片段，使用我们所设计的末端鉴定引物进行末端测序。我们设计了一种新的噬菌体末端鉴定策略，使用该策略鉴定出PaP1基因组DNA具有1190bp长的末端冗余。
3.噬菌体PaP1基因组的生物信息学及比较基因组学分析。通过GC含量的分析确定PaP1基因组的复制起始位点位于该基因组末端。通过开放阅读框（ORF）和编码序列（CDS）的鉴定确定了PaP1基因组有169个推定的基因（其中包含12个tRNA基因）。使用BlastP分析了PaP1基因的推定功能，并将PaP1基因组的注释信息提交给GenBank（登录号：NC_019913）。对铜绿假单胞菌的四株肌尾噬菌体（PaP1、JG004、PAK_P1和vB_PaeM_C2-10_Ab1）进行了比较基因组学分析，构建了其主要衣壳蛋白的进化树，并建立了噬菌体的一个新属：PaP1样噬菌体属。
4.噬菌体PaP1结构蛋白的SDS-PAGE及质谱鉴定。采用浓缩后的PaP1粗制颗粒进行SDS-PAGE实验，将PaP1的结构蛋白显示在聚丙烯酰胺凝胶上。分别切离不同的PaP1蛋白条带，并进行蛋白的胶内酶解，然后进行HPLC-MS。最后一共鉴定出12个PaP1结构蛋白的编码基因，即通过实验证实了这12个PaP1基因的功能是编码结构蛋白。
5.噬菌体PaP1基因组“鸟枪法”测序难题的研究。把之前PaP1基因组“鸟枪法”测序所获得的43个重叠群（contigs）定位在PaP1全基因组上，发现有约一半的序列是“鸟枪法”所测不出来的。我们阅读文献发现“鸟枪法”测序的宿主菌（大肠杆菌DH5α）可以编码识别修饰碱基的酶EndonucleaseⅤ（EndoⅤ）。而PaP1基因组DNA可以被EndoⅤ所切割，同样条件下“鸟枪法”测序成功的PaP3基因组DNA不可以被EndoⅤ所切割。这为PaP1基因组“鸟枪法”测序难题的解开提供了线索。
6. EndoⅤ介导的细菌免疫机制的研究。我们通过λ-Red重组的方法敲除了大肠杆菌DH5α编码EndoⅤ的基因nfi，构建了突变株DH5α Δnfi。然后使用DH5α Δnfi作为“鸟枪法”克隆建库的宿主菌，重新对PaP1基因组进行“鸟枪法”测序，结果顺利完成了其测序。我们通过单分子实时（SMRT）测序的方法鉴定出PaP1基因组DNA中分布有7557个修饰碱基（包括152个m4C和51个m6A）。据此我们提出细菌通过EndoⅤ排斥噬菌体含有修饰碱基核酸的这样一种新的细菌免疫机制。
综上所述，本文对PaP1长91,715bp的基因组进行了详细的注释，通过比较基因组学的分析建立了铜绿假单胞菌肌尾噬菌体中的一个新的属：PaP1样噬菌体属，解析了PaP1的12个结构蛋白编码基因，探讨了“鸟枪”测序法无法测序的PaP1基因组采用二代测序技术就可以测出全序列的根本原因并提供了解决办法，还揭示了细菌通过EndoⅤ免疫排斥外来侵染核酸、保持物种遗传稳定的免疫新机制。
Pseudomonas aeruginosa is a gram-negative opportunistic pathogen, frequently foundin clinical treatment and resistant to many kinds of antibiotics. Pseudomonas aeruginosa isone of the main pathogens in hospital infection. Phages are viruses specifically infectingbacteria and ubiquitous in the biosphere. Estimations of phage numbers are approximatelytenfold higher than those of bacteria. Phages are structurally simple, gnomically small andeasily cultured, making them constantly become important objects and materials of manybasic vital phenomena. Identification and classification of phages are groundwork in phagestudies. Currently, most of the newly identified phages have been categorized into differentgenera; whereas there are still some phages remain unclassified.
Whole genome sequencing and annotation are important work in phage studies.Shot-gun sequencing method had been the most popular genome sequencing method for along time. Some phages could not be successfully sequenced by shot-gun strategy or thesequencing process had to stop halfway. Early in the year2004, our laboratory encounteredPseudomonas aeruginosa phage PaP1with its genome hard to sequence. At that time,research students of our laboratory sent the PaP1genome DNA to a commercial sequencingcompany to have it sequenced by shot-gun strategy, but it failed. Then they sent it toanother commercial sequencing company to have it sequenced by shot-gun strategy, itfailed again. So the sequencing of PaP1genome has to be postponed.
In the year2008, the second generation sequencing method had spread worldwide. Wesent the PaP1genome DNA to a commercial sequencing company to have it sequenced byRoche/454sequencer. It gives us a whole PaP1genome sequence with a length of91,715bp. In this thesis, we will fully annotate the PaP1genome, perform comparative genomicanalysis of this genome, and extensively analyze the reason behind the PaP1genome hard to sequence by shot-gun sequencing method. The results are as follows:
1. Additional analysis of the common bionomics of Pseudomonas aeruginosa PA1andphage PaP1. We observed the growth characteristics, gram-negative morphology,extracellular matrix and flagella of Pseudomonas aeruginosa PA1. The PaP1particles wereconcentrated by using PEG8000, and purified by ultrafiltration. The morphology of PaP1was observed by transmission electron microscope. The result shows that PaP1have anicoschedra head~69nm in diameter and a contractile tail~139nm in length. This is animportant evidence for that PaP1is a member of myovirus.
2. Identification of the terminal redundancy of phage PaP1. Roche/454sequencingmethod reveals a whole PaP1genome sequence91,715bp in length. The linear dsDNAfeature and the location of the PaP1genome ends were characterized by restriction enzymedigestion analysis. We recovered the3’ and5’ end fragments of the PaP1genome, and thenterminal run-off sequencing of the two terminal fragments were carried out to identify theterminal sequence of the PaP1genome. We designed a new strategy to explore the terminalsequence of the PaP1DNA, and the result shows that the PaP1genome has1190bpterminally redundant regions.
3. Bioinformatic and comparative genomic analyses of the PaP1genome. GC skewanalysis reveals that the replication origin of the PaP1genome locates within the terminallyredundant region. The PaP1genome has169putative genes (including12tRNA genes),based on the analysis of open reading frames and coding sequences. BlastP analysis revealsputative functions of these genes. Both the genome sequence and the annotationinformations were submitted to GenBank under the accession number: NC_019913.Comparative genomic analyses of four Pseudomonas aeruginosa myovirus (PaP1, JG004,PAK_P1and vB_PaeM_C2-10_Ab1) were performed. We constructed the phylogenetic treeof these four phages and proposed a new genus: PaP1-like phages.
4. Identification of phage PaP1structural proteins. SDS-PAGE was used to visualizeeach structural protein of phage PaP1in the gel. At least17proteins with molecular weightsranging from6kDa to80kDa were resolved. Each protein band was then excised forHPLC-MS, permitting the allocation of15protein bands to12corresponding PaP1genes.
5. Analysis of the puzzle of the PaP1genome hard to sequence by shot-gun strategy.Shot-gun sequencing of the PaP1genome only gives us43contigs. We located these contigs to the PaP1genome sequence revealed by Roche/454sequencer and found thatapproximately half of PaP1genome can not be sequenced by shot-gun strategy. Then wefound that EndonucleaseⅤ（EndoⅤ）, produced by E. coli DH5α (the host bacteria ofshot-gun sequencing), can digest the PaP1genome DNA. But with the same condition, thePaP3genome DNA (successfully sequenced by shot-gun strategy) can not be digested byEndo Ⅴ. This result provides a hint for the puzzle of the shot-gun sequencing of the PaP1genome.
6. Analysis of EndoⅤ mediated immunity of bacteria. We knocked out the EndoⅤcoding gene (nfi) of E. coli DH5α using λ-Red recombination system and the recombinantstrain was named E. coli DH5α Δnfi. Then shot-gun sequencing of the PaP1genome DNAwas successfully completed using E. coli DH5α Δnfi as the host bacterium. SMRTsequencing revealed7557modified bases (including152m4C and51m6A) in the PaP1genome. These results indicate that the well-known DNA repair protein EndoⅤ ofE. colican mediate the immunity of modified bases containing invasive phage DNA.
In conclusion, this work fully annotated the PaP1genome, constructed a new genusnamed “PaP1-like phages” within the Myoviridae family, identified12PaP1structuralprotein coding genes, and reveal a new immunity mechanism for bacteria to resist phageDNA.
引文
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